The major goal of this reentry application is to facilitate Dr. Patrick Bilder?s reengagement with science. Dr. Bilder has experienced a recent two-and-a-half year diversion from his career path due to illness. The scientific component of the re-entry application, which aims at characterizing the cell envelope constituents of a set of Mycobacterium tuberculosis (Mtb) mutants with the ability to enhance macrophage tumor necrosis factor (TNF) production, derives from the results generated by an on-going P01 program, on which I am the PI of Project 3. A major component of Project 3 of the P01 Program is to develop effective TB vaccines by targeting Mtb TNFregulating genes to generate mutants with enhanced immunogenecity. This strategy is based on the ability of TNF to promote apoptosis and phagolysosomal fusion, two cellular processes known to augment immunogenicity of an immunological reaction by enhancing cross-priming of cytotoxic CD8 T cells and antigen presentation; respectively. Using a genetic screen, we have identified a set of Mtb genes whose disruption results in mutants with TNF-upregulating phenotype in infected macrophages. In line with the above-stated properties of TNF and validating our vaccine development strategy, these mutants promote apoptosis and phagolysosomal fusion in macrophages and imunization of C57BL/6 mice with these strains elicit a Th1 response superior to that engendered by the wildtype (WT) Mtb strain H37Rv. These TNF-upregulating mutants are disrupted for genes that have been annotated to encode functions capable of modulating the Mtb cell envelope components. The overall goal of this application is to test the hypothesis that the cell envelope of the TNF-upregulating Mtb mutants are different than that of the WT strain, thus resulting in altered interaction with host macrophages, leading to enhanced TNF production by the phagocytes upon infection. To ensure the success of Dr. Bilder?s re-entry into science, we have in place a plan that incorporates bench experience, career development opportunities, multiple venues for scientific intearctions, and didactic courses; in addition, a well established TB investigators with experience pertinent to the subject matter of the proposal -- Dr. Jordi Torrelles (Ohio State University) has been enlisted as Dr. Bilder?s Co-mentor. We believe this proposal will successfully facilitate Dr. Bilder?s re-entry into science and will likely yield information that will shed light on the mechanisms underlying the TNF-enhancing attributes of the Mtb deletion strains, which may, in turn, lead to the development of novel effective anti-TB vaccines.